Our research is directed towards expanding the genetic tools available for the study of cultured mammalian cells and their viruses. The genetic analysis of mammalian tissue culture cells has been quite difficult, in part because of the lack of a stringent conditional lethal system. For this reason we are attempting to isolate nonsense suppressors in cultured mammalian cells. The nonsense suppressor system would be valuable in the analysis of mammalian cells in culture and their viruses because it should be non-leaky and because it will permit direct biochemical identification of the mutant gene product. For this purpose we have isolated, after mutagenesis, 500 independent cell lines lacking hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity. Among these mutants we have identified cell lines containing ochre (UAA), amber (UAG) and opel (UGA) mutations within the HGPRT structural gene. We are now in the process of screening revertants derived from these prepared from mammalian cell extracts and programmed with bacteriophage and mammalian messenger RNAs. These in vitro systems do respond to suppressor tRNA isolated from bacteria and yeast. The HGPRT mutants also afford an opportunity to study selective degradation of abnormal proteins in mammalian cells. These studies will be extended using both biochemical and genetic approaches.